Interpretive Summary: Huanglongbing (HLB) and Asiatic citrus canker (ACC) are bacterial diseases that seriously threaten the Florida citrus industry. Sweet orange and grapefruit are highly susceptible to ACC and improvement through conventional plant breeding is a long-term process, making transgenic solutions attractive. No strong HLB-resistance has been identified within cultivated citrus scion types, so creation of transgenic citrus resistant to HLB and ACC may help maintain viable U.S. citrus production where these diseases are widespread. Broad-spectrum antimicrobial peptides (AMPs) have been the focus for current work, and identification of safe and effective transgenes is essential. Lab tests were conducted to evaluate how 44 AMPs affected the ACC bacterium and two bacterial species related to the unculturable HLB bacterium. Eighteen AMPs previously identified in organisms were tested and several inhibited growth of all three test bacterial species at very low levels. An additional 26 synthetic AMPs were designed based on structures of the most effective AMPs and seven of these suppressed growth of all three test bacteria at very low levels. Most AMPs were comparable in effectiveness across the three bacterial species. Most AMPs showed negligible potential for mammalian toxicity in a lab test. Highly effective AMPs from plant or synthetic sources, with no apparent toxicity, will be produced by genetic engineering into citrus and then resulting plants will be tested for HLB and ACC resistance.

Technical Abstract:
Huanglongbing (HLB, associated with Candidatus Liberibacter sp.) and Asiatic citrus canker (ACC, causal organism Xanthomonas citri subsp. citri (XCC)) are bacterial diseases that seriously threaten sustainability of the Florida citrus industry. Sweet orange and grapefruit are highly susceptible to ACC and improvement through conventional breeding is a long-term process, making transgenic solutions attractive. No strong HLB-resistance has been identified within cultivated citrus scion types: creation of transgenic citrus that would permit economic citrus production where HLB is endemic is a high priority. Little is known about the HLB pathosystem and thus broad-spectrum antimicrobial peptides (AMPs) have been the focus for current work, and identification of safe and effective transgenes is essential to our efforts. In vitro assessment of minimum inhibitory concentration (MIC) for 44 AMPs was conducted using Sinorhizobium meliloti and Agrobacterium tumefaciens as surrogates for the unculturable Liberibacter spp. as they are closely related alpha proteobacteria. XCC is also a gram-negative bacterium and was included in these analyses, in anticipation that HLB and ACC resistance can be achieved with the same AMP transgene, if expressed using non-tissue-specific promoters. Twenty AMPs from diverse sources were initially tested. AMPs with the lowest MICs included tachyplesin 1 from horseshoe crab, SMAP-29 from sheep, D4E1 and D2A21 (which are synthetic AMPs derived through evaluation of critical amino acid residues in AMPs, overall peptide structure, and AMP effectiveness), the human LL-37, and the bee venom AMP melittin. These AMPs inhibited growth of all three test bacterial species at 1 µM or less. An additional 20 synthetic AMPs were designed based on structures of the most effective AMPs and seven of these showed effectiveness at 1 µM or less across all three test bacteria. Most AMPs were comparable in effectiveness across the three bacterial species, but some species x AMP interactions were observed. Hemolytic activity was assessed by exposure of porcine erythrocytes to the AMPs. Hemolysis from most AMPs was not significantly different from water, while melittin (from bee venom) was highly hemolytic.